Nicotine contained in tobacco is considered to be the primary reinforcing component responsible for tobacco addiction in human smokers. The positive effects of nicotine involve the brain's mesolimbic reward pathways whereas the aversive effects appear to be mediated by the habenulo-interpeduncular pathway. Defining the mechanisms that regulate the motivational signals to consume nicotine is essential to understanding the components that contribute to tobacco addiction in human smokers. The PI's past experiences have defined the function of the habenulo-interpeduncular pathway with respect to inhibition of intake at higher doses of nicotine. Through this research, the PI has developed and validated a mouse model for nicotine self-administration, learned molecular techniques for genotyping and assessing mRNA expression in tissue, and assisted with the development and validation of lentiviral vectors, all of which provide a basis for the research proposed. The immediate goals of this proposal are twofold: (1) to further define the mechanisms of the habenulo-interpeduncular pathway and its afferents/efferents that regulate an inhibitory motivational signal to limit nicotine consumption, and (2) to provide an essential training experience for career and professional development to ensure success of the PI as an independent academic scientist. With regard to the first goal, the PI will apply the in vivo mouse neurooptogenetic technique to more clearly define the specific role of the pathways, neurotransmitters and receptors involved in an inhibitory motivational signal. The light-activated inhibitory halorhodopsin pump (eNpHR3.0) will be injected into select brain regions to permit transfection of cholinergic neurons/axons in BAC transgenic choline acetyltransferase (ChAT): Cre mice. Subsequently, the effect of light-mediated activation of this pump on intravenous nicotine self-administration behavior will be examined. It should be noted that this proposal is highly innovative by combining two novel techniques - mouse intravenous nicotine self-administration and optogenetics - to define the neuronal processes regulating nicotine's actions. The PI will receive further assistance/training from Dr. Garret Stuber, Dr. Peter Kalivas, and Dr. David Fitzpatrick. Neurotransmitter and receptor involvement in signal processing and protein changes with acute and chronic nicotine self-administration will also be examined. The overall hypothesis of this study is that the posterior septum transmits an inhibitory motivational signal t the medial habenula (MHb) and that this signal is then relayed to the interpeduncular nucleus (IPN) and then to the dorsal tegmental nucleus (DTN) to inhibit behavioral self-administration of nicotine. It is further hypothesized that cholinergic neurons expressing the ?5 nicotinic acetylcholine receptor (nAChR) subunit modulate information processing within the IPN-DTN pathway and that information is subsequently relayed from the DTN to the ventral tegmental area. Experiments outlined in this proposal will test these hypotheses and may thereby provide valuable insights into the mechanisms underlying the nicotine habit in human tobacco smokers. Further, the excellent environment at The Scripps Research Institute will provide the equipment, space, facilities, and scientific support necessary to complete the studies for the K99 phase. With this training, the PI may then begin to address long-term goals of elucidating the mechanisms by which nicotine modulates brain function and by which neuronal activity alters behavior to promote or inhibit further consumption of the drug. As such, R01 grant submissions during the independent phase may focus on other nAChR subunits and signaling mechanisms (dopamine, serotonin, enkephalin, etc.) in the MHb-IPN pathway for their involvement in nicotine reinforcement, dependence and withdrawal. These findings may then provide a basis for further identification of novel drug targets with the goal of developing more efficacious smoking cessation therapeutics. With regard to career development, the mentored career training in this proposal will be equally essential to ensure the PI's future success as an independent research investigator. First, the PI will obtain didactic instruction by attending a relevant course at eithr Cold Spring Harbor or Woods Hole and will conduct intensive reading of the literature to be discussed with the mentor and/or advisory committee members. Second, the PI will be trained in laboratory management and grant preparation and management. In addition to writing a complete and thorough R01 grant proposal, this training will promote knowledge of the preparation of budgets, tracking of expenditures, maintaining direction and preparation of progress reports. Further instruction will be obtained by attending an NIH Regional Seminar on Program Funding and Grants Administration offered by the NIH Office of Extramural Research. Third, the interactions with the mentor and newly-formed advisory committee will provide necessary feedback regarding progress and presentation skills, as well as input relative to job searches and transitioning to independence. These individuals have a great wealth of knowledge of the drug addiction field, hold numerous R01 and other grants, serve on NIH and non-NIH grant review committees, serve as editors on journals, and have been involved in faculty hiring. In addition, the PI will attend career development seminars offered by the Career and Postdoctoral Services office and Network for Women in Science at Scripps Florida, and will enroll in the Responsible Conduct of Research course. Therefore, with the hands-on, didactic and mentored training components, this plan will substantially enhance the PI's research skills in a new technique (optogenetics), permit her to use this technique under novel, innovative conditions (in conjunction with mouse self-administration), and advance her professional knowledge to ensure success and scientific independence to attain R01 funding in the early independent years as tenure-track faculty. PUBLIC HEALTH RELEVANCE: Tobacco addiction has a significant negative impact on the health and economic status of the individual and society. The proposed research will investigate the role of discrete brain pathways and neurotransmitter systems in mediating an inhibitory signal to nicotine that limits intake of the drug. Together, these data may reveal fundamental insights into the neurobiological mechanisms underlying nicotine reinforcement and dependence, and importantly, may lead to the development of novel therapeutics efficacious for smoking cessation in humans.